Hepatic insulin action: Role of the pentose cycle. Insulin, after binding to its liver receptor, initiates the activation of a signaling effector chain, via PI3-K/Akt activation, and subsequent mTOR activation, stimulates glycolysis, lipogenesis, and via mTOR, glucose-6-P dehydrogenase (G6PDH) and pentose phosphate pathway (PPP) flux. Our data shows that pentose cycle activity, measured using stable isotope flux phenotyping, is not just a passive response to PI3-K/mTOR activation. In the whole body Pten hetero-deficient mouse , basal glucose and insulin are unchanged, glucokinase (GK) is downregulated in the basal state, PPP flux, and glucose/glucose-6-P flux re-cycling, is not increased in the fasted to fed transition, basal hepatic glucose production is unchanged and excess accumulation of hepatic triglycerides (TGs) does not occur. This is in contrast to the hepatic specific Pten KO, which is characterized by basal hypoglycemia and hepatic steatosis. GK expression is thought to be an indicator of hepatic PI3-K action, so these "paradoxical" responses for GK, PPP flux and TG accumulation for the Pten mouse suggest that regulation of glucose/glucose-6-P cycling, and PPP flux, is important for glucose homeostasis. We contend, and will show that the PPP flux is a vital link in the metabolite feedback control of mTOR/Akt/AMPK signaling. Insulin stimulated PI3-K/Akt activity is known to inhibit HGP, p-oxidation and TG secretion via phosphorylation and translocation to the cytoplasm of nuclear Foxol and FoxA2, and GK. Glycolytic flux induction, and fatty acid (FA) synthesis, is dependent upon the nuclear translocation of SREBP-1c and ChREBP. SREBP-1c and ChREBP synergistically induce lipogenic enzyme expression, and the activation of ChREBP is dependent on the amount of xylulose-5-P (X5P) generated in the PPP. NADPH generated in the PPP is crucial for fatty acid and TG synthesis, and additional metabolite feedback to insulin signaling can be postulated to result from phosphatidate and diacylglycerol generated during TG synthesis. The purpose of this grant is to understand how Pten affects signaling feedback between Akt, AMPK and mTOR, how these signals regulate transcription factor activation/translocation in the fasted and fed states, and finally, how these fasted/fed signaling events are dependent on intra-organ metabolite feedback from the hepatic PPP and glycolytic/TG synthesis pathways, and inter-organ flux regulatory mechanisms.